In the last five years, dozens of new genes have been identified that increase or decrease the risk of autoimmune diseases. Many laboratories are now trying to determine how certain autoimmune-predisposing or -protective haplotypes (i.e. combinations of gene polymorphisms) affect the expression, splicing, and function of the encoded protein. However, the current approach, based on studying primary cells from individuals carrying different haplotypes, often gives unclear answers. This grant will try to validate a novel approach to functional genetics of autoimmunity, which is complementary to that for primary cells. Our strategy is to assess functional differences between autoimmune-predisposing and protective genetic variations by directly studying full-length gene haplotypes transfected in human cells. The full-length gene will be carried on a bacterial artificial chromosome (BAC) and manipulated before transfection in order to carry the desired variations. The result will be a series of cell clones that carry different full-length gene haplotypes. The clones will then be subjected to a variety of functional studies. We already have a prototype of the system in-hand, and here we apply for the funding needed to validate our approach and optimize it for large-scale application. Thus we will focus on two well-known autoimmunity genes relevant for T cell function, PTPN22 and IL2RA, and use Jurkat T cells as a model cell line. In Aim 1 we will use our prototype assay to assess the functional effect of gene variations in PTPN22 and IL2RA. In Aim 2 we will experiment with long-PCR-based approaches in the attempt to achieve cloning of haplotypes into BACs directly from patient genomic DNA. This will eliminate the need for gene manipulation in order to obtain the desired haplotypes. Our approach is novel and simple, and does not require collection of primary cells. Once validated and optimized, it can be applied to all autoimmunity genes using a variety of cell lines, and even broadly applied to functional genetics studies of complex human diseases.